Erythropoietin (EPO)
Overview Erythropoietin (EPO) is a glycoprotein hormone that is used for red blood cell proliferation within the bone marrow. EPO is made in the kidneys in which it travels to the bone marrow to bind to the erythropoietin receptors (EpoR), causing a signaling cascade for red blood cell growth. EPO is created by highly specialized epithelial-like cells in the kidney. Darbepoetin alfa is available through the pharmaceutical market as an EPO replacement for people with specific medical conditions. Although athletes have used recombinant EPO to gain a competitive edge in endurance sports by increasing their oxygen carrying capacity, synthetic EPO like Darbepoetin alfa are intended for people that suffer from Anemia. For example, If someone were to have renal disease, there is a possibility of becoming anemic because the body is not producing enough EPO for the red blood cell demand. Darbepoetin alfa is listed on the market as Aranesp®. Derbepoetin alfa is also named Novel Erythropoiesis Stimulating Protein (NESP) and has a better effect for anemia treatments as opposed to recombinant human EPO (rHuEPO). Recombinant DNA Construct for Expression Darbepoetin alfa is a 165 Amino acid protein that differs from EPO because of five amino acid sequence changes. The expression due to this sequence alteration is two more N-linked oligosaccharide chains that allows for enhanced glycosylation. These additional oligosaccharide chains can permit more sialic acid residues, which gives Darbepoetin alfa a lower binding affinity and a longer half-life than EPO or other Erythropoiesis-Stimulating Agents (ESA's). This is an advantage because the dosage as well as the treatment frequency can be decreased. Cells and Expression Darbepoetin alfa is derived from Chinese Hamster ovary cells (CHO cells), which happen to be a widely used mammalian cell for the production of therapeutic recombinant proteins. CHO cells are used in many protein synthesis scenarios because they are compliant to genetic modification, which allows for foreign DNA uptake. The uptake of the DNA allows for the CHO cells to express the desired protein in big quantities. This is why the pharmaceutical industry has more than 20 FDA approved drugs that are developed via CHO cell biomedical technology. EPO is a cytokine used for erythrocyte signaling. The erythropoietin receptor is bound by the molecule, which triggers a signal transduction cascade that controls proliferation and differentiation of precursor cells. This signaling cascade is called the Janus Kinase 2 Signal Transducer and Activator of transcription (JAK2-STAT). Protein Purification A major reason why CHO cells are used for desired protein synthesis is because the extracted proteins can be purified very efficiently. CHO cell DNA contamination in one dose of a given protein product does not exceed picogram levels. Purification is done by chromatography and filtration methods in order to produce the maximum amount of product. The typical process for human recombinant EPO (such as Darbepoetin alfa) is conducted as follows: # Affinity Chromatography for protein capturing # Anion Exchange Chromatography # Reverse-Phase Chromatography # Cation Exchange Chromatography # Size exclusion Chromatography # Nanofiltration # Ultrafiltration (can occur before step 1, 2, or 5) References 1. Scientific Discussion (of Darbepoetin Alfa) 2. Darbepoetin Alfa (Princeton University Research) 3. Drug Bank 4. Erythropoietin (Wikipedia) 5. CHO Consortium 6. Darbepoetin alfa (Wikipedia) 7.Pharmacology of Darbepoetin alfa (Oxford Journals) 8. Aranesp 9. The Erythropoietin Receptor (The Oncologist) 10. Comparing and Contrasting ESA forms (photo credit)